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Novel peptide sequence ("IQ-tag") with high affinity for NIR fluorochromes allows protein and cell specific labeling for in vivo imaging.

Kelly KA, Carson J, McCarthy JR, Weissleder R - PLoS ONE (2007)

Bottom Line: Here we used phage display to identify a novel peptide sequence with nanomolar affinity for near infrared (NIR) (benz)indolium fluorochromes.The developed peptide sequence ("IQ-tag") allows detection of NIR dyes in a wide range of assays including ELISA, flow cytometry, high throughput screens, microscopy, and optical in vivo imaging.The described method is expected to have broad utility in numerous applications, namely site-specific protein imaging, target identification, cell tracking, and drug development.

View Article: PubMed Central - PubMed

Affiliation: Center for Molecular Imaging Research, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, United States of America.

ABSTRACT

Background: Probes that allow site-specific protein labeling have become critical tools for visualizing biological processes.

Methods: Here we used phage display to identify a novel peptide sequence with nanomolar affinity for near infrared (NIR) (benz)indolium fluorochromes. The developed peptide sequence ("IQ-tag") allows detection of NIR dyes in a wide range of assays including ELISA, flow cytometry, high throughput screens, microscopy, and optical in vivo imaging.

Significance: The described method is expected to have broad utility in numerous applications, namely site-specific protein imaging, target identification, cell tracking, and drug development.

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Related in: MedlinePlus

IQ-tag binds with high affinity to GH680 and permits imaging via fluorescence microscopy and FACs.A. Representative sigmoidal dose response curve of IQ-tag binding to the benzindolium fluorochrome (KD = 0.53±0.12 SD). Bars, SEM. B. Immunodetection of GH680 on a glass slide via IQ-tag phage and anti-M13. Integrated pixel density of TMB substrate deposition via anti-M13-HRP reactions on spot dilutions of GH680. C. beads conjugated to GH680 or D beads conjugated to BSA were incubated with IQ-tag displaying FITC labeled phage and imaged via fluorescence microscopy (20x objective, equal exposure, FITC channel). Insets: Pixel topography renditions of the images from C and D. Average fold FITC intensity comparing dye-conjugated and unconjugated beads is 6.1 (p<10−36, t-test).
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pone-0000665-g004: IQ-tag binds with high affinity to GH680 and permits imaging via fluorescence microscopy and FACs.A. Representative sigmoidal dose response curve of IQ-tag binding to the benzindolium fluorochrome (KD = 0.53±0.12 SD). Bars, SEM. B. Immunodetection of GH680 on a glass slide via IQ-tag phage and anti-M13. Integrated pixel density of TMB substrate deposition via anti-M13-HRP reactions on spot dilutions of GH680. C. beads conjugated to GH680 or D beads conjugated to BSA were incubated with IQ-tag displaying FITC labeled phage and imaged via fluorescence microscopy (20x objective, equal exposure, FITC channel). Insets: Pixel topography renditions of the images from C and D. Average fold FITC intensity comparing dye-conjugated and unconjugated beads is 6.1 (p<10−36, t-test).

Mentions: The dependence on context of IQ-tag binding to GH680 was characterized next. First, we determined the affinity of IQ-tag displayed on phage to immobilized GH680-BSA. The binding curves formed a classic sigmoidal shape indicative of specific binding of the dye to IQ-tag with an average KD of 0.53±0.2 nmol/L. To elucidate whether this binding was specifically due to the presentation of IQ-tag on the phage (Fig. 4A), we cloned the sequence encoding this peptide on the p7 coat proteins located on the opposite end of the phage. Phage expressing N′-p7-AIQSPHFF likewise demonstrated a sigmoidal dose response (r2 = 0.984) with high affinity (Kd 0.53±0.12 nmol/L). In contrast, phage without displayed peptides demonstrated negligible binding. To estimate the stoichiometry of the dye binding to phage displaying IQ-tag, a known concentration of phage was incubated with the dye. This resulted in approximately one fluorochrome per peptide (7.4±2.0, SD).


Novel peptide sequence ("IQ-tag") with high affinity for NIR fluorochromes allows protein and cell specific labeling for in vivo imaging.

Kelly KA, Carson J, McCarthy JR, Weissleder R - PLoS ONE (2007)

IQ-tag binds with high affinity to GH680 and permits imaging via fluorescence microscopy and FACs.A. Representative sigmoidal dose response curve of IQ-tag binding to the benzindolium fluorochrome (KD = 0.53±0.12 SD). Bars, SEM. B. Immunodetection of GH680 on a glass slide via IQ-tag phage and anti-M13. Integrated pixel density of TMB substrate deposition via anti-M13-HRP reactions on spot dilutions of GH680. C. beads conjugated to GH680 or D beads conjugated to BSA were incubated with IQ-tag displaying FITC labeled phage and imaged via fluorescence microscopy (20x objective, equal exposure, FITC channel). Insets: Pixel topography renditions of the images from C and D. Average fold FITC intensity comparing dye-conjugated and unconjugated beads is 6.1 (p<10−36, t-test).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC1919420&req=5

pone-0000665-g004: IQ-tag binds with high affinity to GH680 and permits imaging via fluorescence microscopy and FACs.A. Representative sigmoidal dose response curve of IQ-tag binding to the benzindolium fluorochrome (KD = 0.53±0.12 SD). Bars, SEM. B. Immunodetection of GH680 on a glass slide via IQ-tag phage and anti-M13. Integrated pixel density of TMB substrate deposition via anti-M13-HRP reactions on spot dilutions of GH680. C. beads conjugated to GH680 or D beads conjugated to BSA were incubated with IQ-tag displaying FITC labeled phage and imaged via fluorescence microscopy (20x objective, equal exposure, FITC channel). Insets: Pixel topography renditions of the images from C and D. Average fold FITC intensity comparing dye-conjugated and unconjugated beads is 6.1 (p<10−36, t-test).
Mentions: The dependence on context of IQ-tag binding to GH680 was characterized next. First, we determined the affinity of IQ-tag displayed on phage to immobilized GH680-BSA. The binding curves formed a classic sigmoidal shape indicative of specific binding of the dye to IQ-tag with an average KD of 0.53±0.2 nmol/L. To elucidate whether this binding was specifically due to the presentation of IQ-tag on the phage (Fig. 4A), we cloned the sequence encoding this peptide on the p7 coat proteins located on the opposite end of the phage. Phage expressing N′-p7-AIQSPHFF likewise demonstrated a sigmoidal dose response (r2 = 0.984) with high affinity (Kd 0.53±0.12 nmol/L). In contrast, phage without displayed peptides demonstrated negligible binding. To estimate the stoichiometry of the dye binding to phage displaying IQ-tag, a known concentration of phage was incubated with the dye. This resulted in approximately one fluorochrome per peptide (7.4±2.0, SD).

Bottom Line: Here we used phage display to identify a novel peptide sequence with nanomolar affinity for near infrared (NIR) (benz)indolium fluorochromes.The developed peptide sequence ("IQ-tag") allows detection of NIR dyes in a wide range of assays including ELISA, flow cytometry, high throughput screens, microscopy, and optical in vivo imaging.The described method is expected to have broad utility in numerous applications, namely site-specific protein imaging, target identification, cell tracking, and drug development.

View Article: PubMed Central - PubMed

Affiliation: Center for Molecular Imaging Research, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, United States of America.

ABSTRACT

Background: Probes that allow site-specific protein labeling have become critical tools for visualizing biological processes.

Methods: Here we used phage display to identify a novel peptide sequence with nanomolar affinity for near infrared (NIR) (benz)indolium fluorochromes. The developed peptide sequence ("IQ-tag") allows detection of NIR dyes in a wide range of assays including ELISA, flow cytometry, high throughput screens, microscopy, and optical in vivo imaging.

Significance: The described method is expected to have broad utility in numerous applications, namely site-specific protein imaging, target identification, cell tracking, and drug development.

Show MeSH
Related in: MedlinePlus